Raav-based compositions and methods for treating amyotrophic lateral sclerosis

ABSTRACT

The invention relates to inhibitory nucleic acids and rAAV-based compositions, methods and kits useful for treating Amyotrophic Lateral Sclerosis.

RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. § 120 of U.S. patentapplication Ser. No. 16/295,621, filed Mar. 7, 2019, which is acontinuation under 35 U.S.C. § 120 of U.S. patent application Ser. No.15/126,688, filed Sep. 16, 2016, which is a National Stage Applicationof PCT/US2015/021321, filed Mar. 18, 2015, which claims the benefitunder 35 U.S.C. § 119(e) of the filing date of U.S. Provisional PatentApplication Ser. No. 61/955,189, filed Mar. 18, 2014, each of whichapplications is incorporated in their entirety herein by reference.

REFERENCE TO A SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 4, 2021, isnamed U012070061US05-SUBSEQ-JOB and is 14 kilobytes in size.

FIELD OF THE INVENTION

The invention relates to methods and compositions for treating geneticdisease, such as Amyotrophic Lateral Sclerosis.

BACKGROUND OF THE INVENTION

Amyotrophic lateral sclerosis (ALS) is a progressive, generally fatalmotor neuron disorder that sometimes develops concurrently withfrontotemporal dementia (FTD). ALS is encountered in both sporadic(SALS) and familial (FALS) forms. About 10% of cases are transmitted asautosomal dominant traits. An FDA-approved therapy for ALS is riluzole,a compound that prolongs survival by about 10%.

SUMMARY OF THE INVENTION

Aspects of the disclosure relate to compositions and methods formodulating expression of genes associated with amyotrophic lateralsclerosis (ALS). In particular, inhibitory nucleic acids are providedthat are useful for silencing of genes, such as C9orf72 and SOD1, whichare associated with ALS. For example, in some aspects of the disclosureinhibitory nucleic acids are provided that target all variants ofC9orf72. In other aspects of the disclosure, inhibitory nucleic acidsare provided that target a subset of variants of C9orf72. Someembodiments of the disclosure relate to a recognition that, althoughcertain inhibitory nucleic acids, such as miRNAs, generally function inthe cytoplasm, they can be loaded onto Argonaut protein (e.g., AGO2, thecatalytic component of RNA induced silencing complex or RISC) in thecytoplasm and imported back into the nucleus where they can silencepre-mRNA. Thus, in some embodiments, inhibitory nucleic acids (e.g.,miRNAs) are provided that are capable of targeting both the RNA withinthe nucleus and the RNA in the cytoplasm to prevent or inhibiting RNAfunction, including protein translation.

Aspects of the disclosure relate to treatment methods that utilizeintrathecal delivery of AAVs engineered to express inhibitory nucleicacids that silence genes, such as C9orf72 and SOD1, which are associatedwith ALS. In some embodiments, methods are provided for deliveringnucleic acids that utilize neurotropic AAVs, such as AAV9 and AAV.Rh10,to target CNS tissue. The use of AAVs harboring nucleic acids that areengineered to express inhibitory nucleic acids is advantageous in partbecause it overcomes deficiencies associated with having tore-administer non-expressed inhibitory nucleic acids, such as, e.g.,siRNA duplexes and antisense oligonucleotides, since the rAAV episomeswill continually express the inhibitory nucleic acids (e.g., miRNA).Moreover, in some embodiments, methods provided herein are advantageousbecause they allow for the use of relatively low doses of AAVs forsilencing genes in the CNS and minimize the exposure of peripheraltissues to the AAVs.

In other aspects of the disclosure, transgenic mice are provided thatcontain a C9orf72 G₄C₂ expansion. In some embodiments, the modelfacilitates assessment of inhibitory nucleic acids for C9orf72 genesilencing in vitro as well as in vivo in a mammalian CNS. In otheraspects, the use of RAN-translated peptides is disclosed as markers,e.g., for C9orf72 activity. In some embodiments, the transgenic mousemodel facilitates assessment of persistence in the CNS of neurotrophicAAVs, such as AAVs harboring an Rh10 capsid. In some embodiments, thetransgenic mouse model facilitates assessment of incipientimmunogenicity following administration, e.g., via intrathecal delivery.

In some aspects, the disclosure provides a method of inhibiting C9orf72expression in a cell, the method comprising delivering to the cell aninhibitory nucleic acid that targets both pre-mRNA and mRNA encoded by aC9orf72 gene.

In some embodiments, the cell expresses C9orf72 having G₄C₂ expansionsof up to 50, up to 90, up to 160, or up to 200 repeats. In someembodiments, the level of a mRNA encoding isoform B of C9orf72 in thecell is greater than the level of a mRNA encoding isoform A of C9orf72protein in the cell.

In some embodiments, the cell is a cell of the central nervous system.In some embodiments, the cell is a neuron.

In some embodiments, prior to being exposed to the inhibitory nucleicacid, the cell contains intranuclear G₄C₂ foci. In some embodiments,delivery of the inhibitory nucleic acid to the cell results in areduction in intranuclear G₄C₂ foci.

In some embodiments, prior to being exposed to the inhibitory nucleicacid, the cell contains C9 RAN proteins. In some embodiments, deliveryof the inhibitory nucleic acid to the cell results in a reduction in C9RAN protein levels.

In some embodiments, the cell is in vivo. In some embodiments, the cellis in vitro. In some embodiments, the cell is of a subject having one ormore symptoms of FTD or ALS. In some embodiments, the cell is of asubject suspected of having FTD or ALS.

In some aspects, the disclosure provides a method of inhibiting C9orf72expression in the central nervous system (CNS) of a subject, the methodcomprising administering to the CNS of the subject an inhibitory nucleicacid that targets an RNA encoded by the C9orf72 gene, wherein theinhibitory nucleic acid is a microRNA.

In some aspects, the disclosure provides a method of inhibiting C9orf72expression in the central nervous system (CNS) of a subject, the methodcomprising administering to the CNS of the subject an inhibitory nucleicacid that targets both pre-mRNA and mRNA encoded by a C9orf72 gene.

In some embodiments, the inhibitory nucleic acid is a microRNA.

In some embodiments, the step of administering the inhibitory nucleicacid to the subject comprises administering to the subject a recombinantadeno-associated virus (rAAV) harboring a nucleic acid that isengineered to express the inhibitory nucleic acid in a cell of thesubject.

In some aspects, the disclosure provides a method of treating a subjecthaving or suspected of having FTD or ALS, the method comprisingadministering to the subject an effective amount of a recombinantadeno-associated virus (rAAV) harboring a nucleic acid that isengineered to express, in a cell of the subject, an inhibitory nucleicacid that targets both pre-mRNA and mRNA encoded by a C9orf72 gene.

In some embodiments, the rAAV targets CNS tissue. In some embodiments,the rAAV comprises an AAV.Rh10 or AAV9 capsid protein.

In some embodiments, the inhibitory nucleic acid comprises a region ofcomplementarity that is complementary with at least 5 consecutivenucleotides within exon 3 of C9orf72. In some embodiments, the at least5 consecutive nucleotides are within nucleotides 220 to 241 of C9orf72.

In some embodiments, the inhibitory nucleic acid targets mRNA encodingisoform A and mRNA encoding isoform B of C9orf72 protein. In someembodiments, the inhibitory nucleic acid targets C9orf72 variants V1(NM_145005.6; SEQ ID NO: 18), V2 (NM_018325.3; SEQ ID NO: 19), and V3(NM_001256054.1; SEQ ID NO: 20). In some embodiments, the inhibitorynucleic acid targets C9orf72 variants V1 (NM_145005.6; SEQ ID NO: 18)and V3 (NM_001256054.1; SEQ ID NO: 20), but not V2 (NM_018325.3; SEQ IDNO: 19). In some embodiments, the inhibitory nucleic acid targetsC9orf72 variant V1 (NM_145005.6; SEQ ID NO: 18), but not V2(NM_018325.3; SEQ ID NO: 19) and V3 (NM_001256054.1; SEQ ID NO: 20).

In some embodiments, the inhibitory nucleic acid reduces levels ofC9orf72 mRNA in a cell by at least 50%. In some embodiments, theinhibitory nucleic acid reduces levels of C9orf72 pre-mRNA in a cell byat least 50%.

In some embodiments, the inhibitory nucleic acid comprises 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutivenucleotides of a sequence as set forth in any one of SEQ ID NOs: 1 to 8.In some embodiments, the rAAV is administered by intrathecally,intracerebrally, intraventricularly or intravenously.

In some aspects, the disclosure provides a method of inhibiting SOD1expression in a cell, the method comprising delivering to the cell anmiRNA that targets SOD1 mRNA, wherein the miRNA comprises 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotidesof a sequence as set forth in:

SEQ ID NO: 15: AGCATTAAAGGACTGACTGAA (SOD-miR-103); SEQ ID NO: 16:GACTGAAGGCCTGCATGGATT (SOD-miR-117); or SEQ ID NO: 17:CTGCATGGATTCCATGTTCAT (SOD-miR-127).

In some aspects, the disclosure provides a method of treating a subjecthaving or suspected of having ALS, the method comprising administeringto the subject an effective amount of a recombinant adeno-associatedvirus (rAAV) harboring a nucleic acid that is engineered to express, ina cell of the subject, an miRNA that targets RNA encoded by a SOD1 gene.

In some embodiments, the miRNA comprises 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of a sequenceas set forth in:

SEQ ID NO: 15: AGCATTAAAGGACTGACTGAA (SOD-miR-103); SEQ ID NO: 16:GACTGAAGGCCTGCATGGATT (SOD-miR-117); or SEQ ID NO: 17:CTGCATGGATTCCATGTTCAT (SOD-miR-127).

In some embodiments, the rAAV targets CNS tissue. In some embodiments,the rAAV comprises an AAV.Rh10 or AAV9 capsid protein.

In some aspects, the disclosure provides a synthetic microRNA comprisinga sequence as set forth in any one of SEQ ID NOs: 1 to 8.

In some aspects, the disclosure provides a synthetic microRNA comprisinga sequence as set forth as SEQ ID NO: 15: AGCATTAAAGGACTGACTGAA(SOD-miR-103); SEQ ID NO: 16: GACTGAAGGCCTGCATGGATT (SOD-miR-117); orSEQ ID NO: 17: CTGCATGGATTCCATGTTCAT (SOD-miR-127). In some embodiments,the synthetic microRNA further comprise flanking regions of miR-155.

In some aspects, the disclosure provides recombinant nucleic acidencoding the microRNA as set forth in any one of SEQ ID NO: 1 to 8 orSEQ ID NO: 15 to 17 and comprising an inverted terminal repeats (ITR) ofan AAV serotype. In some embodiments, the AAV serotype is selected fromthe group consisting of: AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8,AAV9, AAVRh10, AAV11 and variants thereof.

In some embodiments, the recombinant nucleic acid further comprises apromoter operably linked with a region(s) encoding the microRNA. In someembodiments, the promoter is a tissue-specific promoter. In someembodiments, the promoter is a polymerase II promoter, such as a β-actinpromoter. In some embodiments, the promoter is a polymerase IIIpromoter, such as a U6 promoter.

In some aspects, the disclosure provides a composition comprising arecombinant nucleic acid as described by the disclosure.

In some aspects, the disclosure provides a recombinant Adeno-AssociatedVirus (AAV) harboring a recombinant nucleic acid as described by thedisclosure. In some embodiments, the recombinant AAV further comprisesone or more capsid proteins of one or more AAV serotypes selected fromthe group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV.Rh10, AAV11 and variants thereof.

In some aspects, the disclosure provides a composition comprising arecombinant AAV as described by the disclosure. In some embodiments, thecomposition further comprises a pharmaceutically acceptable carrier.

In some aspects, the disclosure provides a kit comprising a containerhousing a composition as described by the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B provide a diagram of C9orf72 gene and primers. FIG. 1Arepresents the genomic organization of the gene; the lower panels arepre-mRNA variants 1-3. The boxes represent exons and the lines areintrons. The hexanucleotide repeat expansion (red diamond) istranscribed in variants 1 and 3. The ATG start codon and TAA stop codonare as shown. A horizontal line represents the open reading frame foreach variant (note that variants 2 and 3 produce the same protein). FIG.1A shows the locations of the TaqMan primers used to distinguish thepre-mRNA isoforms; FIG. 1B shows the three spliced mRNAs are shown;figure annotations as above with the positions of the primer pairs thatdetect the three different spliced mRNA isoforms.

FIG. 2 shows qRT-PCR data relating to detection of C9orf72 variants.TaqMan probe assays designed to detect each individual variant (V₁, andV₃) or all variants (V_(all)) mRNA were tested on various cell lines aswell as human brain tissue. The results are means±SD from 3 biologicalreplicates.

FIG. 3 shows in vitro data relating to miRNA-mediated knockdown of humanC9orf72. Relative levels of C9orf72 mRNA after knockdown of C9orf72 withartificial C9miR-220. This miRNA is located in exon 3 and targets allvariants. These are results from 3 biological replicates of mir220 (CBApromoter-GFP) transient transfections in HEK293T cells. The control is amiR against SOD1.

FIG. 4. shows in vitro data relating to knockdown of C9orf72 mRNA andpre-mRNA. C9-miR220 is an artificial miRNA designed to target bases220-241 of the ORF of C9orf72. This miRNA binds in exon 3 thus targetingall mRNA variants. The miRNA was cloned into either a plasmid using a U6or Chicken Beta-Actin promoter and transfected into Hek-293 cells. RNAwas extracted 72 hours after transfection and DNAse treated prior to RT.Quantitative RT-PCR was done using custom TaqMan probes that eitherdetect spliced (e.g., V_(all) mRNA) or unspliced (e.g., V_(all)pre-mRNA) variants. The results are means±SD from 3 biologicalreplicates.

FIG. 5 shows a Southern analysis of DNA (Left panel). Lanes: (A) ALSlymphoblast DNA RB8088. C9orf72 expanded, ˜8 kb (900 repeats); (B) ALScortex DNA RB9783. C9orf72 expanded, ˜9 kb (1000 repeats); (C) ALScortex DNA RB2952. C9orf72, wild type, no expansion band; (D) Mousespleen DNA Non transgenic; (E): Mouse spleen DNA CH523-111K12_523.C9orf72 expansion BAC, ˜4.5 kb (350 repeats). For each specimen, 25 ugof DNA was digested by Xbal and separated on 0.8% agar gel 2.5 hours at80-V. Hybridization was at 55oc with an RNA oligo probe (G₄C₂)4-DIG andvisualized with CDP-Star. Right: Hybridization of hippocampus of WT andC9orf72 mice with G₄C₂-CyA probe for sense strand of RNA.

FIGS. 6A-6D provide data indicating robust EGFP transduction and miRNAmediated knockdown in the spinal cord after I.T. injection withrAAV.Rh10. A 4 year-old male marmoset was I.T. injected with either arAAV.Rh10.CBEGFP or rAAV.Rh10.U6ant—SOD1 miR at a dose of 5×10e12GCs/kg. The animal was necropsied 2 weeks later and CNS tissues isolatedand fixed. Forty micron sections of spinal cord were stained withantibody against EGFP and visualized by DAB. All sections werecounterstained with Haematoxylin. Shown are low magnification images (4×objective) of (FIG. 6A) Lumbar spinal cord, (FIG. 6B) thoracic spinalcord, and (FIG. 6C) cervical spinal cord. FIG. 6D shows motor neuronswere laser captured from the lumbar spinal of control (rAAV.Rh10.GFP)and treated (rAAV.Rh10.GFP-miR-SOD1) marmosets and assayed for levels ofmiR-SOD1 microRNA and SOD1 mRNA RT-qPCR.

FIGS. 7A-7B illustrate rAAV Vector design for miRNA-mediated silencingof C9orf72. FIG. 7A shows the targeting miRNA sequences are cloned intoa stem-loop, which is flanked by pri-miRNA sequences of miR-155. FIG. 7Bshows the sequence targeting the C9-miRs are then cloned into pro-viralAAV plasmids with either a polymerase II (chicken B-actin) or polymeraseIII (U6) promoters for in vitro testing and rAAV packaging.

FIG. 8 illustrates qRT-PCR data relating to different C9orf72 variantsin mice.

FIG. 9 illustrates that C9orf72 mutant transgenic mice develop poly(GP)inclusions in the frontal cortex.

FIGS. 10A-10B show antibodies directed against RAN-translated peptidesdetect inclusions in C9FTD/ALS brain tissue. FIG. 10A shows specificityof antibodies was confirmed by Western blot analysis using lysates fromcells transfected to express GFP-tagged (GA)5, (GR)5, (GP)5, (PA)5 or(PR)5. FIG. 10B shows Anti-GA, anti-GR and anti-GP immunoreactiveinclusions are detected throughout the brain of C9FTD/ALS, including inthe cerebellum, as shown here.

FIGS. 11A-11C show rAAV vector design for miRNA mediated silencing ofSOD1. The flanking regions of miR-155 were cloned upstream of the BGHpoly A region of a proviral AAV expression cassette composed of the CMVenhancer, chicken Beta actin hybrid promoter with a short SV40 intron.FIG. 11A shows the sequence targeting the hSOD1 mRNA was cloned into themiR-155 backbone. FIG. 11B shows two tandem copies of this miRNA werecloned into a vector that expresses either GFP or vector that onlyexpresses the miRNAs, as would be desired in the clinical setting. FIG.11C shows an alignment of the human, Rhesus and Marmoset SOD1 genesequences showing that the mature miR-SOD1-127 is targeting a sequencethat is 100% conserved among the primates.

FIGS. 12A-12B show data relating to in vivo rAAV mediated knockdown ofhuman SOD1 in the transgenic mice. Transgenic mice expressing the humansSOD1 G93A mutation were injected with rAAV9 vectors expressing theanti-SOD1 miRs. FIG. 12A shows newborn mice were administered 1.0×10¹²particles of either a GFP control vector or one expressing theSOD1miR-127 via the facial vein. Mice were sacrificed 4 weeks afterdelivery and the muscle was analyzed for total hSOD1 expression byquantitative real-time RT-PCR. FIG. 12B shows adult mice were injectedwith 5×10¹⁰ vector particles directly into the striatum. Mice weresacrificed 3 weeks after injection and the brain tissue was analyzed forhSOD1 expression by quantitative real-time RT-PCR.

FIG. 13 shows data relating to AAV.Rh10 vector constructs (top), andresults indicating reduction of SOD1 expression in marmoset liver(bottom).

FIG. 14 outlines assays performed to assess silencing of SOD1 inmarmosets.

FIG. 15 is an overview of assays conducted in marmosets.

FIG. 16 shows that in 3 male marmosets injected intrathecally (IT) andsubjected to laser capture micro-dissection (LCM) (animals 6, 8, 9 inFIG. 15), SOD1 expression was reduced in MNs by AAV.Rh10 CB-2x-miR-SOD1(light grey) and U6-miR-SOD1 (dark gray). The U6 promoter drove higherlevels of the anti-SOD1 miR and more effectively silenced SOD1.

FIGS. 17A-17B show data related to 3 male marmosets (animals 6, 8, 9 inFIG. 15), FIG. 17A shows SOD1 expression was reduced in MNs and non-MNsby IT AAV.Rh10 CB-2x -miR-SOD1 (light grey) and U6-miR-SOD1 (dark gray).The U6 construct produced greater knock-down. FIG. 17B shows relativeGFP expression in MNs and non-MNs by IT rAAV.Rh10 CB-2x-mir-SOD1 andU6-miR-SOD1. The U6 construct produced the highest GFP expression.

FIG. 18 shows that in the same 3 male marmosets described in FIGS.17A-17B, the IT injection of AAV.Rh10 CB-2x-miR-SOD1 (light grey) andU6-miR-SOD1 (dark gray) also produced silencing of SOD1 in the lowerbrainstem. In these studies, qPCR was performed on whole tissuehomogenates (not laser captured neurons).

FIG. 19 shows data relating to an assessment of motor neuron (MN) SOD1expression using RNA hybridization (RNAScope) in cords of two malemarmosets (#2 and 3 in FIG. 15). In #2, IT injection with CB-GFP (nomiR) achieved some GFP expression in MNs (ChAT pos) which showedprominent SOD1 expression (left). In #3, IT injection with U6-SOD1miR-CB-GFP produced GFP expression in MN and reduction in SOD1expression in the same neurons (right).

FIG. 20 shows data relating to treatment of G93A SOD1 mice withCB-miR-SOD1 vector. Mice were intravenously injected with 2×10¹² gc ofvector (CB-GFP or CB-miR-SOD1-GFP) at day 56-68 of age and subsequentlyblindly monitored until advanced paralysis required euthanasia. Resultsshow median survival was 108 days for the CB-GFP group and 130 days forthe CB-miR-SOD1-GFP group (log-rank test, p=0.018), indicating asignificant increase in survival of CB-miR-SOD1-treated mice.

DETAILED DESCRIPTION

Aspects of the disclosure relate to compositions and methods formodulating expression of genes associated with amyotrophic lateralsclerosis (ALS). Aspects of the disclosure relate to improved genetherapy compositions and related methods for treating ALS using therecombinant adeno-associated viral (rAAV) vectors. In particular, rAAVsare provided that harbor nucleic acids engineered to express inhibitorynucleic acids that silence genes, such as C9orf72 and SOD1, which areassociated with ALS. In some embodiments, the disclosure utilizes arecombinant AAV (e.g., rAAV.Rh10) to deliver a microRNA to the CNS andthereby silence an ALS gene, such as SOD1 or C9orf72.

ALS occurs in both familial (FALS) and sporadic (SALS) forms. Asignificant number of FALS cases are associated with expansions of anon-coding hexanucleotide G₄C₂ expansion in the gene C9orf72. Theseexpansions are also detected in 10-20% of familial frontotemporaldementia (FTD), 10% of sporadic FTD and in ˜5% of SALS. These statisticsdefine the C9orf72 G₄C₂ expansion as a common cause of ALS. In normalindividuals, the G₄C₂ expansion ranges in size from 2 or 3 to upwards of25 repeats; by contrast, FTD/ALS patients have hundreds or eventhousands of these repeats. Transcription from the normal C9orf72 geneyields three mRNA variants V₁ (e.g., Genbank: NM_145005.6; SEQ ID NO:18), V₂ (e.g., Genbank: NM_018325.3; SEQ ID NO: 19), and V₃ (e.g.,Genbank: NM_001256054.1; SEQ ID NO: 20). Transcript V₁ contains exons1a-6b and codes for a 222 amino acid protein. Exons V₂ and V₃respectively contain exons 2-12 and exons 1b-12 and code for the same481aa protein (FIG. 1).

Aspects of the disclosure relate to a recognition that V₁ and V₃ harborthe G₄C₂ expansion. Analysis of human ALS and FTD brains with thisexpansion has shown intranuclear accumulation of the RNA transcripts,generating RNA intranuclear foci in the frontal cortex and spinal cord.This supports that the expansion in transcripts V₁ and V₃ is a primaryadverse agent causing cytotoxicity in motor neurons. While the functionsof the proteins encoded by C9orf72 are not well characterized,bioinformatics approaches indicate that the C9orf72 protein sharesstructural features with (DENN) and GDP/GTP exchange factors (GEF)4 andso may regulate membrane cell trafficking among other potentialfunctions. Cytotoxicity of the G₄C₂ expansions may be associated withone or more gain-of-function mechanisms, such as, for example: 1)excessive sequestering by the RNA foci of transcription factors (likemuscleblind in myotonic dystrophy); 2) repeat-associated non-ATG (RAN)translation of the expanded repeat, leading to expression of dipeptides(Gly-Ala; Gly-Pro; Gly-Arg); the peptides produced in this fashion formneuronal inclusions throughout the CNS; (3) and induction ofhaploinsufficiency due to decreased C9orf72 transcript expression.Aspects of the disclosure relate to the use of rAAV (e.g.,intrathecally-delivered rAAV.Rh10) to introduce an inhibitory nucleicacid (e.g., a microRNA) to silence expression of the transcripts ofC9orf72 that harbor the offending G₄C₂ expansion. In some embodiments,the disclosure provides methods and compositions that achieve silencekey pre-mRNA and mature mRNA transcripts of C9orf72 in vitro.

Mutations in the gene encoding Superoxide dismutase (SOD1), located onchromosome 21, have been linked to familial amyotrophic lateralsclerosis. Superoxide dismutase (SOD1) is an enzyme encoded by the SOD1gene. SOD1 binds copper and zinc ions and is one of three superoxidedismutases responsible for destroying free superoxide radicals in thebody. The encoded isozyme is a soluble cytoplasmic and mitochondrialintermembrane space protein, acting as a homodimer to convert naturallyoccurring, but harmful, superoxide radicals to molecular oxygen andhydrogen peroxide.

Frequent SOD1 mutations that occur and cause ALS include A4V, H46R andG93A. Typically, these ALS-causing SOD1 mutations act in a dominantfashion, such that a single mutant copy of the SOD1 gene may besufficient to cause the disease. It is believed that they mutationsresult in a toxic gain of function as the mutant enzymes typicallyretain enzymatic activity. Accordingly, mutant SOD1 can cause a widerange of cellular defects including mitochondrial dysfunctions,oxidative stress, calcium misregulation, aggregation of aberrantlyprocessed proteins, endoplasmic reticulum (ER) stress, axonal transportdisruption, neurotransmitter misregulation, programmed cell death andinflammation. Aspects of the disclosure relate to the use of rAAV (e.g.,rAAV.Rh10) to introduce an inhibitory nucleic acid (e.g., a microRNA)into cells to silence expression of mutant SOD1.

Inhibitory Nucleic Acids

In some embodiments, the disclosure provides inhibitory nucleic acidsthat inhibit expression of genes that cause ALS, such as SOD1 andC9orf72. In some embodiments, the inhibitory nucleic acid is a nucleicacid that hybridizes to at least a portion of the target nucleic acid,such as an RNA, pre-mRNA, mRNA, and inhibits its function or expression.In some embodiments, the inhibitory nucleic acid is single stranded ordouble stranded. In some embodiments, the inhibitory nucleic acid is amicroRNA (miRNA). In some embodiments, the inhibitory nucleic acid is amicroRNA comprising a targeting sequence having flanking regions ofmiR-155.

In some embodiments, the inhibitory nucleic acid is 5 to 30 bases inlength (e.g., 10-30, 15-25, 19-22). The inhibitory nucleic acid may alsobe 10-50, or 5-50 bases length. For example, the inhibitory nucleic acidmay be one of any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 bases inlength. In some embodiments, the inhibitory nucleic acid comprises orconsists of a sequence of bases at least 80% or 90% complementary to,e.g., at least 5, 10, 15, 20, 25 or 30 bases of, or up to 30 or 40 basesof, the target nucleic acid, or comprises a sequence of bases with up to3 mismatches (e.g., up to 1, or up to 2 mismatches) over 10, 15, 20, 25or 30 bases of the target nucleic acid.

In some embodiments, any one or more thymidine (T) nucleotides oruridine (U) nucleotides in a sequence provided herein may be replacedwith any other nucleotide suitable for base pairing (e.g., via aWatson-Crick base pair) with an adenosine nucleotide. For example, T maybe replaced with U, and U may be replaced with T. In some embodiments,inhibitory nucleic acids are provided that inhibit expression of genesin a cell of the central nervous system. In some embodiments, the cellis a neuron, astrocyte, or oligodendrocyte.

In some embodiments, the cell expresses C9orf72 having G₄C₂ expansionsof up to 50, up to 90, up to 160, up to 200, up to 300, up to 400, up to500 repeats, up to 600 repeats or more. In some embodiments, theinhibitory nucleic acid comprises a sequence as set forth in any one ofSEQ ID NOs: 1 to 8. In some embodiments, the level of a mRNA encodingisoform B of C9orf72 in the cell is greater than the level of a mRNAencoding isoform A of C9orf72 protein in the cell. In some embodiments,the cell contains detectable levels of intranuclear G₄C₂ foci. In someembodiments, the cell contains detectable levels of C9 RAN proteins.

In some embodiment, the cell expresses a mutant SOD1enzyme. In someembodiments, the SOD1 mutation is selected from: A4V, H46R and G93A. Insome embodiments, the inhibitory nucleic acid comprises or consists of asequence as set forth as SEQ ID NO: 15: AGCATTAAAGGACTGACTGAA(SOD-miR-103); SEQ ID NO: 16: GACTGAAGGCCTGCATGGATT (SOD-miR-117); orSEQ ID NO: 17: CTGCATGGATTCCATGTTCAT (SOD-miR-127).

Methods of Use

Methods are provided herein for inhibiting the expression of genes thatare associated with FTD and/or ALS, such as C9orf72 or SOD1. In someembodiments, methods are provided for inhibiting the expression ofC9orf72 in a cell that involve delivering to the cell an inhibitorynucleic acid that targets both pre-mRNA and mRNA encoded by a C9orf72gene. In some embodiments, methods are provided for inhibiting theexpression of C9orf72 in a cell that involve administering to the CNS ofthe subject an inhibitory nucleic acid that targets an RNA encoded bythe C9orf72 gene, wherein the inhibitory nucleic acid is a microRNA.

In some embodiments, methods are provided for inhibiting C9orf72expression in the central nervous system (CNS) of a subject. In someembodiments, the methods involve administering to the CNS of the subjectan inhibitory nucleic acid that targets both pre-mRNA and mRNA encodedby a C9orf72 gene. In some embodiments, the subject has or is suspectedof having FTD or ALS. In some embodiments, the methods involveadministering to the subject an effective amount of a recombinantadeno-associated virus (rAAV) harboring a nucleic acid that isengineered to express, in a cell of the subject, an inhibitory nucleicacid that targets both pre-mRNA and mRNA encoded by a C9orf72 gene. Insome embodiments, the inhibitory nucleic acid comprises a sequence asset forth in any one of SEQ ID NOs: 1 to 8.

In some embodiments, methods are provided for inhibiting SOD1 expressionin a cell. In some embodiments, the methods involve delivering to thecell an miRNA that targets SOD1 mRNA, wherein the miRNA comprises 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutivenucleotides of a sequence as set forth in:

SEQ ID NO: 15: AGCATTAAAGGACTGACTGAA (SOD-miR-103);

SEQ ID NO: 16: GACTGAAGGCCTGCATGGATT (SOD-miR-117); or

SEQ ID NO: 17: CTGCATGGATTCCATGTTCAT (SOD-miR-127), or of acomplementary sequence of any one of them. In some embodiments, the SOD1mRNA is set forth in GenBank: EF151142.1.

In some embodiments, methods are provided for treating a subject havingor suspected of having ALS. In some embodiments, the methods involveadministering to the subject an effective amount of a recombinantadeno-associated virus (rAAV) harboring a nucleic acid that isengineered to express, in a cell of the subject, an miRNA that targetsRNA encoded by a SOD1 gene.

In accordance with the foregoing, certain methods provided hereininvolve administering to a subject an effective amount of a recombinantAdeno-Associated Virus (rAAV) harboring any of the recombinant nucleicacids disclosed herein. In general, the “effective amount” of a rAAVrefers to an amount sufficient to elicit the desired biologicalresponse. In some embodiments, the effective amount refers to the amountof rAAV effective for transducing a cell or tissue ex vivo. In otherembodiments, the effective amount refers to the amount effective fordirect administration of rAAV to a subject. As will be appreciated bythose of ordinary skill in this art, the effective amount of therecombinant AAV of the invention varies depending on such factors as thedesired biological endpoint, the pharmacokinetics of the expressionproducts, the condition being treated, the mode of administration, andthe subject. Typically, the rAAV is administered with a pharmaceuticallyacceptable carrier.

In some instances, after administration of the rAAV at least oneclinical outcome parameter or biomarker (e.g., intranuclear G₄C₂ RNAfoci, RAN-protein expression, etc.) associated with the FTD or ALS isevaluated in the subject. Typically, the clinical outcome parameter orbiomarker evaluated after administration of the rAAV is compared withthe clinical outcome parameter or biomarker determined at a time priorto administration of the rAAV to determine effectiveness of the rAAV.Often an improvement in the clinical outcome parameter or biomarkerafter administration of the rAAV indicates effectiveness of the rAAV.Any appropriate clinical outcome parameter or biomarker may be used.Typically, the clinical outcome parameter or biomarker is indicative ofthe one or more symptoms of an FTD or ALS. For example, the clinicaloutcome parameter or biomarker may be selected from the group consistingof: intranuclear G₄C₂ RNA foci, RAN-protein expression, SOD1 expression,C9orf72 expression, memory loss, and presence or absence of movementdisorders such as unsteadiness, rigidity, slowness, twitches, muscleweakness or difficulty swallowing, speech and language difficulties,twitching (fasciculation) and cramping of muscles, including those inthe hands and feet.

Recombinant AAVs

In some aspects, the invention provides isolated AAVs. As used hereinwith respect to AAVs, the term “isolated” refers to an AAV that has beenisolated from its natural environment (e.g., from a host cell, tissue,or subject) or artificially produced. Isolated AAVs may be producedusing recombinant methods. Such AAVs are referred to herein as“recombinant AAVs”. Recombinant AAVs (rAAVs) may have tissue-specifictargeting capabilities, such that a transgene of the rAAV is deliveredspecifically to one or more predetermined tissue(s). The AAV capsid isan important element in determining these tissue-specific targetingcapabilities. Thus, a rAAV having a capsid appropriate for the tissuebeing targeted can be selected. In some embodiments, the rAAV comprisesa capsid protein having an amino acid sequence corresponding to any oneof AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11and variants thereof. The recombinant AAVs typically harbor anrecombinant nucleic acid of the invention.

Methods for obtaining recombinant AAVs having a desired capsid proteinare well known in the art (See, for example, U.S. Patent PublicationNumber 2003/0138772, the contents of which are incorporated herein byreference in their entirety). AAV capsid proteins that may be used inthe rAAVs of the invention a include, for example, those disclosed in G.Gao, et al., J. Virol, 78(12):6381-6388 (June 2004); G. Gao, et al, ProcNatl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); US 2003-0138772, US2007/0036760, US 2009/0197338, and WO 2010/138263, the contents of whichrelating to AAVs capsid proteins and associated nucleotide and aminoacid sequences are incorporated herein by reference. Typically themethods involve culturing a host cell which contains a nucleic acidsequence encoding an AAV capsid protein or fragment thereof; afunctional rep gene; a recombinant AAV vector composed of AAV invertedterminal repeats (ITRs) and a transgene; and sufficient helper functionsto permit packaging of the recombinant AAV vector into the AAV capsidproteins.

Suitable AAVs that may be used in the methods provided herein aredisclosed in U.S. Patent Publication Number 2013/0195801, entitled “CNSTARGETING AAV VECTORS AND METHODS OF USE THEREOF,” and published on Aug.1, 2013; and U.S. Patent Publication Number 2012/0137379, entitled“NOVEL AAV'S AND USES THEREOF,” and published on May 31, 2012. Thecontents of these publications are incorporated herein by reference forall purposes.

The components to be cultured in the host cell to package a rAAV vectorin an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e.g.,recombinant AAV vector, rep sequences, cap sequences, and/or helperfunctions) may be provided by a stable host cell which has beenengineered to contain one or more of the required components usingmethods known to those of skill in the art. Most suitably, such a stablehost cell will contain the required component(s) under the control of aninducible promoter. However, the required component(s) may be under thecontrol of a constitutive promoter. Examples of suitable inducible andconstitutive promoters are provided herein. In still anotheralternative, a selected stable host cell may contain selectedcomponent(s) under the control of a constitutive promoter and otherselected component(s) under the control of one or more induciblepromoters. For example, a stable host cell may be generated which isderived from 293 cells (which contain E1 helper functions under thecontrol of a constitutive promoter), but which contain the rep and/orcap proteins under the control of inducible promoters. Still otherstable host cells may be generated by one of skill in the art.

The recombinant AAV vector, rep sequences, cap sequences, and helperfunctions required for producing the rAAV of the invention may bedelivered to the packaging host cell using any appropriate geneticelement (vector). The selected genetic element may be delivered by anysuitable method, including those described herein. The methods used toconstruct any embodiment of this invention are known to those with skillin nucleic acid manipulation and include genetic engineering,recombinant engineering, and synthetic techniques. See, e.g., Sambrooket al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virionsare well known and the selection of a suitable method is not alimitation on the present invention. See, e.g., K. Fisher et al, J.Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, recombinant AAVs may be produced using the tripletransfection method (e.g., as described in detail in U.S. Pat. No.6,001,650, the contents of which relating to the triple transfectionmethod are incorporated herein by reference). Typically, the recombinantAAVs are produced by transfecting a host cell with a recombinant AAVvector (comprising a transgene) to be packaged into AAV particles, anAAV helper function vector, and an accessory function vector. An AAVhelper function vector encodes the “AAV helper function” sequences(e.g., rep and cap), which function in trans for productive AAVreplication and encapsidation. In some embodiments, the AAV helperfunction vector supports efficient AAV vector production withoutgenerating any detectable wild-type AAV virions (e.g., AAV virionscontaining functional rep and cap genes). Non-limiting examples ofvectors suitable for use with the present invention include pHLP19,described in U.S. Pat. No. 6,001,650 and pRep6cap6 vector, described inU.S. Pat. No. 6,156,303, the entirety of both incorporated by referenceherein. The accessory function vector encodes nucleotide sequences fornon-AAV derived viral and/or cellular functions upon which AAV isdependent for replication (e.g., “accessory functions”). The accessoryfunctions include those functions required for AAV replication,including, without limitation, those moieties involved in activation ofAAV gene transcription, stage specific AAV mRNA splicing, AAV DNAreplication, synthesis of cap expression products, and AAV capsidassembly. Viral-based accessory functions can be derived from any of theknown helper viruses such as adenovirus, herpesvirus (other than herpessimplex virus type-1), and vaccinia virus.

In some aspects, the invention provides transfected host cells. The term“transfection” is used to refer to the uptake of foreign DNA by a cell,and a cell has been “transfected” when exogenous DNA has been introducedinside the cell membrane. A number of transfection techniques aregenerally known in the art. See, e.g., Graham et al. (1973) Virology,52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual,Cold Spring Harbor Laboratories, New York, Davis et al. (1986) BasicMethods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousnucleic acids, such as a nucleotide integration vector and other nucleicacid molecules, into suitable host cells.

A “host cell” refers to any cell that harbors, or is capable ofharboring, a substance of interest. Often a host cell is a mammaliancell. A host cell may be used as a recipient of an AAV helper construct,an AAV minigene plasmid, an accessory function vector, or other transferDNA associated with the production of recombinant AAVs. The termincludes the progeny of the original cell which has been transfected.Thus, a “host cell” as used herein may refer to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

In some aspects, the invention provides isolated cells. As used hereinwith respect to cell, the term “isolated” refers to a cell that has beenisolated from its natural environment (e.g., from a tissue or subject).As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants. As used herein, the terms“recombinant cell” refers to a cell into which an exogenous DNA segment,such as DNA segment that leads to the transcription of abiologically-active polypeptide or production of a biologically activenucleic acid such as an RNA, has been introduced.

As used herein, the term “vector” includes any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors. In some embodiments, useful vectors arecontemplated to be those vectors in which the nucleic acid segment to betranscribed is positioned under the transcriptional control of apromoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrases“operatively positioned,” “under control” or “under transcriptionalcontrol” means that the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene. The term “expression vector orconstruct” means any type of genetic construct containing a nucleic acidin which part or all of the nucleic acid encoding sequence is capable ofbeing transcribed. In some embodiments, expression includestranscription of the nucleic acid, for example, to generate abiologically-active polypeptide product or inhibitory RNA (e.g., shRNA,miRNA) from a transcribed gene.

The foregoing methods for packaging recombinant vectors in desired AAVcapsids to produce the rAAVs of the invention are not meant to belimiting and other suitable methods will be apparent to the skilledartisan.

Recombinant AAV Vectors

The recombinant nucleic acids of the invention may be recombinant AAVvectors. The recombinant AAV vector may be packaged into a capsidprotein and administered to a subject and/or delivered to a selectedtarget cell. “Recombinant AAV (rAAV) vectors” are typically composed of,at a minimum, a transgene and its regulatory sequences, and 5′ and 3′AAV inverted terminal repeats (ITRs). The transgene may comprise, asdisclosed elsewhere herein, one or more regions that encode one or moreinhibitory nucleic acids (e.g., miRNAs) comprising a nucleic acid thattargets an endogenous mRNA of a subject. The transgene may also comprisea region encoding an exogenous mRNA that encodes a protein (e.g., afluorescent protein).

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)). The ITR sequences are about 145 bp in length. In someembodiments, substantially the entire sequences encoding the ITRs areused in the molecule, although some degree of minor modification ofthese sequences is permissible. The ability to modify these ITRsequences is within the skill of the art. (See, e.g., texts such asSambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., ColdSpring Harbor Laboratory, New York (1989); and K. Fisher et al., JVirol., 70:520 532 (1996)). An example of such a molecule employed inthe present invention is a “cis-acting” plasmid containing thetransgene, in which the selected transgene sequence and associatedregulatory elements are flanked by the 5′ and 3′ AAV ITR sequences. TheAAV ITR sequences may be obtained from any known AAV, includingpresently identified mammalian AAV types.

Thus, the recombinant nucleic acids may comprise inverted terminalrepeats (ITR) of an AAV serotypes selected from the group consisting of:AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV.Rh10, AAV11 andvariants thereof. The recombinant nucleic acids may also include apromoter operably linked with the one or more first inhibitory RNAs, theexogenous mRNA, and/or the one or more second inhibitory RNAs. Thepromoter may be tissue-specific promoter, a constitutive promoter orinducible promoter.

In addition to the major elements identified above for the recombinantAAV vector, the vector also includes conventional control elements whichare operably linked with elements of the transgene in a manner thatpermits its transcription, translation and/or expression in a celltransfected with the vector or infected with the virus produced by theinvention. As used herein, “operably linked” sequences include bothexpression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest. Expression control sequencesinclude appropriate transcription initiation, termination, promoter andenhancer sequences; efficient RNA processing signals such as splicingand polyadenylation (polyA) signals; sequences that stabilizecytoplasmic mRNA; sequences that enhance translation efficiency (e.g.,Kozak consensus sequence); sequences that enhance protein stability; andwhen desired, sequences that enhance secretion of the encoded product. Anumber of expression control sequences, including promoters which arenative, constitutive, inducible and/or tissue-specific, are known in theart and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence) andregulatory sequences are said to be operably linked when they arecovalently linked in such a way as to place the expression ortranscription of the nucleic acid sequence under the influence orcontrol of the regulatory sequences. If it is desired that the nucleicacid sequences be translated into a functional protein, two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably linked to a nucleic acidsequence if the promoter region were capable of effecting transcriptionof that DNA sequence such that the resulting transcript might betranslated into the desired protein or polypeptide. Similarly two ormore coding regions are operably linked when they are linked in such away that their transcription from a common promoter results in theexpression of two or more proteins having been translated in frame. Insome embodiments, operably linked coding sequences yield a fusionprotein. In some embodiments, operably linked coding sequences yield afunctional RNA (e.g., miRNA).

For nucleic acids encoding proteins, a polyadenylation sequencegenerally is inserted following the transgene sequences and before the3′ AAV ITR sequence. A rAAV construct useful in the present inventionmay also contain an intron, desirably located between thepromoter/enhancer sequence and the transgene. One possible intronsequence is derived from SV-40, and is referred to as the SV-40 T intronsequence. Any intron may be from the β-Actin gene. Another vectorelement that may be used is an internal ribosome entry site (IRES).

The precise nature of the regulatory sequences needed for geneexpression in host cells may vary between species, tissues or celltypes, but shall in general include, as necessary, 5′ non-transcribedand 5′ non-translated sequences involved with the initiation oftranscription and translation respectively, such as a TATA box, cappingsequence, CAAT sequence, enhancer elements, and the like. Especially,such 5′ non-transcribed regulatory sequences will include a promoterregion that includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancersequences or upstream activator sequences as desired. The vectors of theinvention may optionally include 5′ leader or signal sequences. Thechoice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer), the SV40 promoter, and the dihydrofolate reductasepromoter. Inducible promoters allow regulation of gene expression andcan be regulated by exogenously supplied compounds, environmentalfactors such as temperature, or the presence of a specific physiologicalstate, e.g., acute phase, a particular differentiation state of thecell, or in replicating cells only. Inducible promoters and induciblesystems are available from a variety of commercial sources, including,without limitation, Invitrogen, Clontech and Ariad. Many other systemshave been described and can be readily selected by one of skill in theart. Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system, the ecdysone insectpromoter, the tetracycline-repressible system, thetetracycline-inducible system, the RU486-inducible system and therapamycin-inducible system. Still other types of inducible promoterswhich may be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly. In another embodiment, the native promoter, or fragment thereof,for the transgene will be used. In a further embodiment, other nativeexpression control elements, such as enhancer elements, polyadenylationsites or Kozak consensus sequences may also be used to mimic the nativeexpression.

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences (e.g., promoters, enhancers, etc.) are well knownin the art. In some embodiments, the promoter is a chicken β-actinpromoter.

In some embodiments, one or more bindings sites for one or more ofmiRNAs are incorporated in a transgene of a rAAV vector, to inhibit theexpression of the transgene in one or more tissues of a subjectharboring the transgenes, e.g., non-liver tissues, non-lung tissues. Theskilled artisan will appreciate that binding sites may be selected tocontrol the expression of a transgene in a tissue specific manner. ThemiRNA target sites in the mRNA may be in the 5′ UTR, the 3′ UTR or inthe coding region. Typically, the target site is in the 3′ UTR of themRNA. Furthermore, the transgene may be designed such that multiplemiRNAs regulate the mRNA by recognizing the same or multiple sites. Thepresence of multiple miRNA binding sites may result in the cooperativeaction of multiple RISCs and provide highly efficient inhibition ofexpression. The target site sequence may comprise a total of 5-100,10-60, or more nucleotides. The target site sequence may comprise atleast 5 nucleotides of the sequence of a target gene binding site.

In some embodiments, the cloning capacity of the recombinant RNA vectormay be limited and a desired coding sequence may involve the completereplacement of the virus's 4.8 kilobase genome. Large genes may,therefore, not be suitable for use in a standard recombinant AAV vector,in some cases. The skilled artisan will appreciate that options areavailable in the art for overcoming a limited coding capacity. Forexample, the AAV ITRs of two genomes can anneal to form head to tailconcatamers, almost doubling the capacity of the vector. Insertion ofsplice sites allows for the removal of the ITRs from the transcript.Other options for overcoming a limited cloning capacity will be apparentto the skilled artisan.

Recombinant AAV Administration

rAAVs are administered in sufficient amounts to transfect the cells of adesired tissue and to provide sufficient levels of gene transfer andexpression without undue adverse effects. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the selected tissue (e.g., livertissue, lung tissue) and administration subcutaneously,intraopancreatically, intranasally, parenterally, intravenously,intramuscularly, intrathecally, intracerebrally, orally,intraperitoneally, by inhalation or by another route. Routes ofadministration may be combined, if desired.

Delivery of certain rAAVs to a subject may be, for example, byadministration into the bloodstream of the subject. Administration intothe bloodstream may be by injection into a vein, an artery, or any othervascular conduit. Moreover, in certain instances, it may be desirable todeliver the rAAVs to brain tissue, meninges, neuronal cells, glialcells, astrocytes, oligodendrocytes, cereobrospinal fluid (CSF),interstitial spaces and the like. In some embodiments, recombinant AAVsmay be delivered directly to the spinal cord or brain (e.g., prefrontalcortex) by injection into the ventricular region, as well as to thestriatum (e.g., the caudate nucleus or putamen of the striatum), andneuromuscular junction, or cerebellar lobule, with a needle, catheter orrelated device, using neurosurgical techniques known in the art, such asby stereotactic injection (see, e.g., Stein et al., J Virol73:3424-3429, 1999; Davidson et al., PNAS 97:3428-3432, 2000; Davidsonet al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. GeneTher. 11:2315-2329, 2000).

In certain circumstances it will be desirable to deliver the rAAV-basedtherapeutic constructs in suitably formulated pharmaceuticalcompositions disclosed herein either intrathecally, intracerebrally,intravenously, subcutaneously, intraopancreatically, intranasally,parenterally, intravenously, intramuscularly, orally, intraperitoneally,or by inhalation.

It can be appreciated by one skilled in the art that desirableadministration of rAAV-based therapeutic constructs can also include exvivo administration. In some embodiments, ex vivo administrationcomprises (1) isolation of cells or tissue(s) of interest from asubject, (2) contacting the cells or tissue(s) with rAAVs in sufficientamounts to transfect the cells or tissue to provide sufficient levels ofgene transfer and expression without undue adverse effect, and (3)transferring cells or tissue back into the subject. In some embodiments,cells or tissues may be cultured ex vivo for several days before and/orafter transfection.

Cells or tissues can be isolated from a subject by any suitable method.For example, cells or tissues may be isolated by surgery, biopsy (e.g.,biopsy of skin tissue, lung tissue, liver tissue, adipose tissue), orcollection of biological fluids such as blood. In some embodiments,cells are isolated from bone marrow. In some embodiments, cells areisolated from adipose tissue. In some embodiments, cells are isolatedfrom a lipoaspirate. Appropriate methods for isolating cells fromadipose tissue for ex vivo transfection are known in the art. See, e.g.,Kuroda, M., et al., (2011), Journal of Diabetes Investigation, 2:333-340; Kouki Morizono, et al. Human Gene Therapy. January 2003, 14(1):59-66; and Patricia A. Zuk, Viral Transduction of Adipose-Derived StemCells, Methods in Molecular Biology, 1, Volume 702, Adipose-Derived StemCells, Part 4, Pages 345-357.

In some embodiments, the isolated cells comprise stem cells, pluripotentstem cells, neuroprogenitor cells, lipoaspirate derived stem cells,liver cells (e.g., hepatocytes), hematopoietic stem cells, mesenchymalstem cells, stromal cells, hematopoietic cells, blood cells,fibroblasts, endothelial cells, epithelial cells, or other suitablecells. In some embodiments, cells to be transfected are inducedpluripotent stem cells prepared from cells isolated from the subject.

In an embodiment, cells or tissue(s) are transduced at a multiplicity ofinfection (MOI) of at least 10 infectious units (i.u.) of a rAAV percell (for example, 10, 100, 1,000, 5,000, 10,000, 100,000 or more i.u.)or at a functionally equivalent viral copy number. In one embodiment,cells or tissue(s) are transduced at a MOI of 10 to 10,000 i.u. Routesfor transfer of transfected cells or tissue(s) into a subject include,but are not limited to, subcutaneously, intraopancreatically,intranasally, parenterally, intravenously, intravascularly,intramuscularly, intrathecally, intracerebrally, intraperitoneally, orby inhalation. In some embodiments, transfected cells are administeredby hepatic portal vein injection. In some embodiments, transfected cellsare administered intravascularly. Methods for ex vivo administration ofrAAV are well known in the art (see, e.g., Naldini, L. Nature ReviewsGenetics (2011) 12, 301-315, Li, H. et al. Molecular Therapy (2010) 18,1553-1558, and Loiler et al. Gene Therapy (2003) 10, 1551-1558).

Recombinant AAV Compositions

The rAAVs may be delivered to a subject in compositions according to anyappropriate methods known in the art. The rAAV, which may be suspendedin a physiologically compatible carrier (e.g., in a composition), may beadministered to a subject, e.g., a human, mouse, rat, cat, dog, sheep,rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, ora non-human primate (e.g., Marmoset, Macaque). The compositions of theinvention may comprise a rAAV alone, or in combination with one or moreother viruses (e.g., a second rAAV encoding having one or more differenttransgenes).

In some embodiments, to assess gene silencing in relatively largeprimates, experiments are performed in African Green Monkeys or otherrelatively large primates. In some embodiments, rAAV vectors expressingmiRNAs (e.g., miR-SOD1) are injected in the CSF of such primates bothcaudally using an IT injection and rostrally using cisterna magnainjections.

Suitable carriers may be readily selected by one of skill in the art inview of the indication for which the rAAV is directed. For example, onesuitable carrier includes saline, which may be formulated with a varietyof buffering solutions (e.g., phosphate buffered saline). Otherexemplary carriers include sterile saline, lactose, sucrose, calciumphosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, andwater. Still others will be apparent to the skilled artisan.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The dose of rAAV virions required to achieve a desired effect or“therapeutic effect,” e.g., the units of dose in vector genomes/perkilogram of body weight (vg/kg), will vary based on several factorsincluding, but not limited to: the route of rAAV administration, thelevel of gene or RNA expression required to achieve a therapeuticeffect, the specific disease or disorder being treated, and thestability of the gene or RNA product. One of skill in the art canreadily determine a rAAV virion dose range to treat a subject having aparticular disease or disorder based on the aforementioned factors, aswell as other factors that are well known in the art. An effectiveamount of the rAAV is generally in the range of from about 10 μl toabout 100 ml of solution containing from about 10⁹ to 10¹⁶ genome copiesper subject. Other volumes of solution may be used. The volume used willtypically depend, among other things, on the size of the subject, thedose of the rAAV, and the route of administration. For example, forintravenous administration a volume in range of 10 μl to 100 μl, 100 μlto 1 ml, 1 ml to 10 ml, or more may be used. In some cases, a dosagebetween about 10¹⁰ to 10¹² rAAV genome copies per subject isappropriate. In some embodiments the rAAV is administered at a dose of10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ genome copies per subject. In someembodiments the rAAV is administered at a dose of 10¹⁰, 10¹¹, 10¹²,10¹³, or 10¹⁴ genome copies per kg.

In some embodiments, rAAV compositions are formulated to reduceaggregation of AAV particles in the composition, particularly where highrAAV concentrations are present (e.g., ˜10¹³ GC/ml or more). Methods forreducing aggregation of rAAVs are well known in the art and, include,for example, addition of surfactants, pH adjustment, salt concentrationadjustment, etc. (See, e.g., Wright F R, et al., Molecular Therapy(2005) 12, 171-178, the contents of which are incorporated herein byreference.)

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens. Typically, these formulations may contain at least about 0.1%of the active ingredient or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active ingredient ineach therapeutically-useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Dispersions may also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. In many cases the form issterile and fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art. For example, one dosage may be dissolvedin 1 ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the host. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual host.

Sterile injectable solutions are prepared by incorporating the activerAAV in the required amount in the appropriate solvent with various ofthe other ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The rAAV compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present invention intosuitable host cells. In particular, the rAAV vector delivered transgenesmay be formulated for delivery either encapsulated in a lipid particle,a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art. Recently, liposomes weredeveloped with improved serum stability and circulation half-times (U.S.Pat. No. 5,741,516). Further, various methods of liposome and liposomelike preparations as potential drug carriers have been described (U.S.Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures. In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs, radiotherapeutic agents, viruses, transcriptionfactors and allosteric effectors into a variety of cultured cell linesand animals. In addition, several successful clinical trials examiningthe effectiveness of liposome-mediated drug delivery have beencompleted.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 .ANG, containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the rAAV may be used.Nanocapsules can generally entrap substances in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use.

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe rAAV compositions to a host. Sonophoresis (e.g., ultrasound) hasbeen used and described in U.S. Pat. No. 5,656,016 as a device forenhancing the rate and efficacy of drug permeation into and through thecirculatory system. Other drug delivery alternatives contemplated areintraosseous injection (U.S. Pat. No. 5,779,708), microchip devices(U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al.,1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) andfeedback-controlled delivery (U.S. Pat. No. 5,697,899).

Kits and Related Compositions

The recombinant nucleic acids, compositions, rAAV vectors, rAAVs, etc.described herein may, in some embodiments, be assembled intopharmaceutical or diagnostic or research kits to facilitate their use intherapeutic, diagnostic or research applications. A kit may include oneor more containers housing the components of the invention andinstructions for use. Specifically, such kits may include one or moreagents described herein, along with instructions describing the intendedapplication and the proper use of these agents. In certain embodimentsagents in a kit may be in a pharmaceutical formulation and dosagesuitable for a particular application and for a method of administrationof the agents. Kits for research purposes may contain the components inappropriate concentrations or quantities for running variousexperiments.

The kit may be designed to facilitate use of the methods describedherein by researchers and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for animal administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may bein the form of a liquid, gel or solid (powder). The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container. The kit may have one ormore or all of the components required to administer the agents to asubject, such as a syringe, topical application devices, or IV needletubing and bag.

Exemplary embodiments of the invention are described in more detail bythe following examples. These embodiments are exemplary of theinvention, which one skilled in the art will recognize is not limited tothe exemplary embodiments.

EXAMPLES Example 1: Assessment and Targeting of C9orf72 Expression

Recombinant adeno-associated viral vectors have been developed thatdeliver miRNAs targeting C9orf72.

Assessment of C9orf72 Expression in Cell Lines and Normal Human Brain:

Two types of cell lines were used that express either WT or mutantC9orf72. A set of 83 lymphoblastoid cells lines were obtained frompatients in 78 familial ALS (FALS) pedigrees that are C9orf72 G₄C₂expansion positive. In addition, continuous HEK and SH-SY5Y cell lineswere generated that have:

-   -   2.0 kb of the C9orf72 promoter upstream of exon 1a,    -   exons 1a and 1b with the intervening intron containing the G₄C₂        repeat, and    -   2.1 kb of the following intron and exon 2, whose start codon        drives luciferase.

Four sub-lines were produced from these cell lines that have G₄C₂expansions of 50, 90, 160 and 200 repeats. Fibroblast cultures were alsoobtained from C9orf72 G₄C₂ expansion cases.

To probe for the principle transcripts of C9orf72 a series of probes andprimers were produced that detect either the pre-mRNA or the splicedmRNA. As depicted in FIG. 1 (top), TaqMan probes were developed for thedifferent pre-mRNA isoforms. One probe, V_(all) detects all pre-mRNAtranscripts, while two others (V₁ or V₃) detect the pre-mRNA isoforms V₁or V₃. As shown in FIG. 1 (bottom), primer pairs were generated thatdetect three distinct spliced mRNA isoforms. V₁ detects isoform B, whileprimer pairs V₂ and V₃ detect two variants of isoforms A.

As shown in FIG. 2, the TaqMan primers do detect the three majorpre-mRNA transcripts from HEK293 and SH-SY5Y cells and from human brain.The transcript levels for V₁ and V₃ are considerably smaller thanV_(all), indicating that, as shown, the predominant transcript in brainand in these cells is V₂.

MicroRNA Mediated Silencing of Expression of Pre-mRNA and Spliced mRNAin Cells:

The ability of a microRNA targeting the C9orf72 gene to silence its RNAtranscripts was assessed. An artificial microRNA, designated C9-miR220that targets bases 220-241 of the ORF in exon 3 of C9orf72 wasdeveloped. Because this miRNA binds in exon 3, it is expected to targetall of the mRNA variants. FIG. 3 shows in vitro miRNA-mediated knockdownof human C9orf72. These are results from 3 biological replicates ofmir220 (CBA promoter-GFP) transient transfections in HEK293T cells. Thecontrol is a miR against SOD1.

The miRNA was cloned into two different plasmids, using either a U6 orthe Chicken Beta-Actin (CBA) promoter. These plasmids were thentransfected into Hek-293 cells. After 72 hours, transcripts were assayedusing quantitative RT-PCR with the custom TaqMan probes that detect thepre-mRNA transcript or with the primers that detect the spliced mRNAvariants. As shown in FIGS. 3 and 4, whether driven by the U6 or the CBApromoter, both forms of the C9-miR220 microRNA reduced levels of splicedmRNA by about 50%. The level of the pre-mRNA transcript was reduced to˜65% (e.g. ˜35% reduction) by the CBA-C9miR220 microRNA, while theU6-C9miR220 suppressed levels of the pre-mRNA to ˜25% (e.g. ˜75%reduction). (See FIG. 4) These results demonstrate silencing both thepre-mRNA and mRNA of the C9orf72 gene using an artificial microRNA.

Generation of Mouse Model with BAC Transgenic G₄C₂ Expansion:

To generate a mouse model of C9orf72-mediated ALS, a bacterialartificial chromosome (BAC) was isolated from cells of patients havingALS with G₄C₂ expansions with ˜580 repeats and 45 repeats. The BAChaving ˜580 repeats spans exons 1-6 of C9orf72 (Hg18chr0:27,561,112-27,714,301), while the BAC having 45 repeats spans thefull coding sequence. Circularized DNA from these BACs was used togenerate transgenic mice. 49 pups were obtained from the 580 repeatBACs, of which 3 were positive for the G₄C₂ expansion by PCR assay. Oneof these three showed germline transmission and produced progeny thathave bred well; a colony of these mice with sustained transmission oftransgene was established. The original founder aged to ˜14 months oldwithout an overt motor neuron phenotype. However, brains of C9 BACtransgenic mice at 4 and 6 months of age and presented with salientfeatures. First, as shown in FIG. 5 (lane E), a Southern blot of genomicDNA isolated from the BAC C9 transgenic mouse reveals a dense,heterogeneous band running roughly from 4.5 to ˜6.0 kb; this compareswell with results using lymphoblastoid (A) and brain (B) DNA from anindividual with a G₄C₂ expansion. No such band is evident in DNA frombrain of an individual without an expansion (lane c) or a non-transgenicmouse (D). A second observation was that probing of sections of thehippocampus from both the 4 and the 6 month 580 repeat BAC C9 transgenicmouse with a G₄C₂-CyA probe (to detect the sense-strand RNA) revealed anabundance of intranuclear RNA foci also present throughout the rest ofthe brain and the spinal cord. (See FIG. 5, right panel). These weredetected by a “blinded” observer. The hippocampus control/WT mouse didnot show these foci. These results indicate: (1) stable transmission ofa BAC C9orf72 transgene with a G₄C₂ expansion; and (2) that the micerecapitulate the intranuclear deposits of sense-strand RNA found inhuman C9orf72 mediated ALS. It has been determined that these foci arenot detected after treatment with RNAse. Because these BAC transgenic C9mice have nuclear RNA foci, silencing of transcript expression fromC9orf72 even in the absence of motor neuron disease can be evaluated byassaying for the presence of foci.

MicroRNA Design:

The miRNA AAV platform is based on miR-155. A stem-loop with a targetingsequence is cloned into the context of the miR-155 flanking regions forefficient recognition and processing by Drosha/DGCR8 complex (FIG. 7).The miRNA design yields a mature 21mer miRNA guide sequence that haseither an adenine or uracil at the 5′ end. The choice of U or A at the5′ end is driven by the fact that the Mid domain of Ago2 interacts withthe 5′ end of the mature miRNA and has a 20-fold higher affinity forthese two bases over cytosine, and guanine. This design also favorsthermodynamic incorporation of the guide strand into the RISC complex.MiRNAs are designed to target areas of low secondary and tertiarycomplexity target mRNA. This is done with RNA folding algorithms withthe goal of increasing the likelihood of miRNA:mRNA cognate binding atthe target site. As shown in FIG. 7B the miRNAs are then cloned into apro-viral plasmid with ITRs expressing GFP and the miRNA of interesteither from a polymerase II or polymerase III promoter. 8 miRNA werecloned into these plasmids that target either variants 1 and 3selectively or all variants (see Table 1).

TABLE 1 miRNAs Cloned to Target C9orf72 Targeting SEQ miRNA ID TargetTarget Name Sequence NO: Variants Exon miR-C9-123 5′-TTTGGAGCCC 1V1-V2-V3 3 AAATGTGCCTT-3′ miR-C9-220 5′-TATAGCACCA 2 V1-V2-V3 3CTCTCTGCATT-3′ miR-C9-220- 5′-TATAGCACCAC 3 V1-V2-V3 3 3′mmTCTCTGCTAA-3′ miR-C9-228 5′-TTTACATCTA 4 V1-V2-V3 3 TAGCACCACTC-3′miR-C9-496 5′-AATACTCTGAC 5 V1-V2-V3 3 CCTGATCTTC-3′ miR-C9-215′-TGACGCACCTC 6 V1-V3 1a-1b TCTTTCCTAG-3′ miR-C9-48 5′-TTTACGTGGG 7V1-V3 1a-1b CGGAACTTGTC-3′ miR-C9-65 5′-TAGATATCAAGCG 8 V1 1a-3TCATCTTT-3′

As shown in Table 1 potential miRNAs have been identified and cloned forthe region spanning the hexanucleotide repeat. Pre-mRNA isoforms V₁ andV₃, were targeted because these encompass the G₄C₂ hexanucleotiderepeat; as in Table 1, mir-C9-21 and -48 are expected to target V₁ andV₃.

In addition, miRNAs were used that target all variants. MiR-C9-220 iseffective for knock down in vitro of both the mRNA and pre-mRNA species.In certain cases, an miRNA with 40-50% knockdown efficiency in vitrotranslates to knockdown of more than 80% in vivo due to the increasedefficiency of transduction and genome copies achieved with a viralvector. This miRNA function in the nucleus as determined by pre-mRNAknockdown. Nuclear targeting can be improved by modifying the last 3bases of 3′ end of the miRNA to be detargeted from the cognate mRNA.When miRNAs are not 100% complementary to their message and aredetargeted at the 3′ they form significantly more stable complexes withAgo2. This would increase the residence time of the miRNA in the Ago2complex and thereby increase the possibility of nuclear translocation.As show in Table 1 we have cloned an miRNA that has a 3′ mismatch(miR-C9-220-3′mm) which is useful for assessing this activity.

In Vitro Knockdown of C9orf72:

HEK-293T cells and SHSY-5Y cells were transiently transfected with JetPrime reagent according to the manufacturer's protocol. Transfection ofpatient fibroblasts uses the protocol for primary fibroblasts on theNucleofactor electroporator (Lonza AG). Cells are collected 48 hourspost transfection, and RNA isolation is performed using Trizol reagent.RNA is then DNAse treated (Turbo DNA-free kit, Applied Biosystems) andreverse-transcribed (High Capacity RNA-to-cDNA kit, Applied Biosystems).For pre-mRNA detection transcript levels are quantified by RT-qPCR (FastSYBR Green mastermix and primer sets mentioned in the Tables 2 and 3below, Applied biosystems). For mRNA detection, transcripts arequantified by RT-qPCR (TaqMan mastermix and TaqMan assays in tablebelow, Applied Biosystems). Expression data is analyzed by the 2^(ΔΔCt).

Primer Design for Pre-mRNA Detection of C9orf72:

Two primer sets were designed for the detection of pre-mRNA. The firstprimer set (V_(all)) detects all variants, because the primers arelocated between exon 2 and the adjacent intron. The second set ofpre-mRNA primers (V₁, V₃) detects variants 1 and 3; the primers arelocated between exon 1 and the adjacent intron (see FIG. 1). Primersequences are shown in the table below:

TABLE 2 C9orf72 pre-mRNA RT-qPCR Assays Name Primer SEQ ID NO:V_(all)-pre- 5′-ACGTAACCTAC  9 mRNA-FP GGTGTCCC-3′ V₁ and V₃-5′-TGCGGTTGCG 10 pre-mRNA-FP GTGCCT-3′ GAPDH-FP 5′-CTCATGACCA 11CAGTCCATGC-3′ V_(all)-pre- 5′-CTACAGGCTG 12 mRNA-RP CGGTTGTTTC-3′V₁ and V₃- 5′-CCACCAGTC 13 pre-mRNA-RP GCTAGAGGCGA-3′ GAPDH-RP5′-ATGACCTTGC 14 CCACAGCCTT-3′Primer-Probe Design for mRNA Detection of C9orf72:

For the detection of spliced mRNA, primer-probe sets were used. Each setspans exon junctions to discriminate from genomic DNA without having toperform a DNase digestion. V₁ detects only variant 1; the primer andprobe set span exons 1a and 3. V₂ spans the junction between exon 2 andexon 3. V₃, which detects variant 3, spans the junction of exon 1b andexon 3. Finally, V_(all) detects all variants; this primer probe setspans the splice junction between exons 3 and 4 (see FIG. 1). TaqManPrimer-probe sequences were ordered through Life Technologies as shownin the table below

TABLE 3 C9orf72 mRNA RT-qPCR Assays Name Catalog# Sequences V₁ 4331182Hs00331877_ml V₂ 4400294 Custom V₃ 4400294 Custom V_(all) 4331182Hs00376619_ml GAPDH 4331182 Hs02758991_glFluorescence In Situ Hybridization (FISH) of G₄C₂ Nuclear Foci:

Detection of G₄C₂ in tissue and patient fibroblasts is achieved byfixing with 4% PFA for 10-20 min on ice, washed 3× with PBS andincubated in 70% Ethanol overnight at 4° C. 40% formamide +2×SSC areadded for 20 min at room temperature. The hybridization buffer (250 ul)is prepared with a Cy3 probe specific for the hexanucleotide expansion(G₄C₂), incubated for 2 hours at 37° C., and then washed with 40%formamide+1×SSC for 30 min at 37° C.; followed by 2 washes with 1×SSC,RT for 15 min. Slides are then mounted and cover slipped withDAPI-containing mounting media (see FIG. 5, right panel).

C9orf72 Quantitative Real-Time PCR:

RNA was extracted from cell using Trizol and \reverse-transcribed (HighCapacity RNA-to-cDNA kit, Applied Biosystems). Following standardprotocols, C9orf72 transcripts levels were quantified by RT-qPCR usingFast Taqman mastermix and the Taqman assays in Tables 2 and 3 (AppliedBiosystems). Relative Quantification was determined using the 2^(−ΔΔct)method.

Quantification of G₄C₂ Nuclear Foci:

The frequency of occurrence of RNA foci in patient fibroblasts areassessed by analyzing random microscopically photographed fields at 60×.Automated counting of RNA foci is carried out using the FishJ algorithmmacro in the ImageJ software.

Statistical Analysis:

Relative expression of C9orf72 transcripts for both mRNA and pre-mRNAafter transfection with the various plasmids are analyzed using the2^(−ΔΔct) equation. Values for at least three biological replicatescomparing controls (GFP-Scramble-miR) to experimental (GFP-C9-miR) wereanalyzed with a two sample t-test for statistical significance. Asecondary endpoint in the experiments involving patient fibroblast wasthe average presence of G₄C₂ nuclear foci. Foci data were obtained fromFishJ digital image analysis for at least 3 biological replicatescomparing controls versus experimental transfections, and again werecompared using a two sample student t-test.

Example 2: In Vivo Efficacy of Intrathecally-Delivered RecombinantAdeno-Associated Virus Type Rh10 (rAAV.Rh10-C9miR) in SilencingExpression of Pre-mRNA and Mature mRNA from the C9orf72 Gene in Mice

Intrathecally delivered rAAV.Rh10 expressing anti-C9 miRs reduce centralnervous system levels of C9orf72 RNA transcripts in both wild-type miceand BAC-derived C9orf72^(mutant) transgenic mice. The effectiveness ofrAAV.Rh10-C9miR in suppressing levels of C9orf72 and the associated G₄C₂transcripts are evaluated in transgenic mice.

Primers V₁, V₂, V₃, and V_(all) are used to assess C9orf72 transcripts.As shown in FIG. 8, assays using V₁, V₂ and V₃ primers detecttranscripts of the transgenic mouse, whereas V_(all) primers detect bothmouse and human C9orf72.

The effective of rAAV.Rh10-C9miR on reducing levels of the pre-mRNA andspliced mRNA transcripts of C9orf72 is evaluated using both wildtype andour C9orf72^(mutant) transgenic mice. The extent to whichrAAV.Rh10-C9miR reduces numbers of RNAi foci in the transgenic mice isevaluated. The extent to which rAAV.Rh10-C9miR reduces levels of c9-RANproteins [poly(G), poly(GA), poly(GR)] is also evaluated.

rAAV.Rh10-C9miRs are administered in wildtype and C9orf72^(mutant)transgenic mice to assess knockdown of the endogenous C9orf72 pre-mRNAand spliced mRNA. C9orf72^(mutant) transgenic mice demonstrate thepresence RNA foci as indicated in FIG. 9, CNS tissue from theC9orf72^(mutant) transgenic mice immunostains positively forRAN-translated peptides. Thus, changes in the occurrence of foci andRAN-translated peptides is used to assess effectiveness ofrAAV.Rh10-C9miRs administration. Reduction in dipeptide levels, forexample, serves as a measure of efficacy of silencing.

rAAV.Rh10-C9miRs in the C9orf72^(mutant) transgenic mice used to assessthe extent to which C9-miRs achieve reductions in (1) pre-mRNA and mRNAlevels; (2) numbers of RNA foci, and (3) production of c9 RAN proteins[poly(G), poly(GA), poly(GR)]. Fluorescence in situ hybridization (FISH)of G₄C₂ nuclear foci is performed on brain and spinal cord tissue oftreated and un-treated mice, as shown in FIG. 5.

As outlined in Tables 4 and 5 rAAVs are injected both neonatal and adultmice; for the former intravenous delivery is used; for the latter, thedelivery is intrathecal. Neonatal injections allow widespread CNStransduction with a small volume of vector. Intrathecal administrationreduces the amount of virus required to transduce the CNS, and itminimizes systemic exposure to the rAAV.

TABLE 4 Wildtype Mouse Studies with AAVRh10-C9-miRs Injection Age inC57BL/6 P1 (Neonate)/ Treatment Dose P1 (Neonate)/Dose P1 (Neonate)/DoserAAV.Rh10- n = 12 (Males), n = 12 (Males), 1 e¹² n = 12 (Males), 5 e¹⁰C9miRs 1.0 e¹¹ vg vg vg rAAV.Rh10- n = 12 (Males), n = 12 (Males), 1 e¹²n = 12 (Males), 5 e¹⁰ Controls 1.0 e¹¹ vg vg vg PBS Injected n = 12(Males), n = 12 (Males), 1 e¹² n = 12 (Males), 5 e¹⁰ Controls 1.0 e¹¹ vgvg vg

TABLE 5 Transgenic Mouse Studies with AAVRh10-C9-miRs Injection Age inC9orf72^(mutant) Transgenic mice Treatment P1 (Neonate) P 28(Neonate)/Dose 3 Months/Dose rAAV.Rh10-C9miRs n = 12 (Males), 1.0 e¹¹ n= 12 (Males), 1 e¹² n = 12 (Males), 5 e¹⁰ vg vg vg rAAV.Rh10-Controls n= 12 (Males), 1.0 e¹¹ n = 12 (Males), 1 e¹² n = 12 (Males), 5 e¹⁰ vg vgvg PBS Injected n = 12 (Males), 1.0 e¹¹ n = 12 (Males), 1 e¹² n = 12(Males), 5 e¹⁰ Controls vg vg vg

Neonate Peripheral Injection:

For neonate injections, hypothermia is used to anesthetize animals priorto intravenous administration. Animals are placed on a bed of wet icefor 1-3 minutes, then injected in the facial vein for P1 and caudal veinfor p28 and returned to bedding with their littermates. The neonateinjection procedure takes approximately 5 minutes.

Lumbar Intrathecal Injection:

Adult mice are anesthetized with isoflurane in an induction chamber at2.5%. Once asleep, the animals are transferred to a nose cone wherecontinuous isoflurane is administered. Mice are injected with of eithera vector encoding a miR against C9orf72 or a scrambled control miR at adose of 5×10¹⁰ vector genomes/animal in a 50 volume. This dose isequivalent to 2×10¹² vg/kg (considering a 25 gr. mouse). Intrathecal(IT) administration is performed using a 30-gauge, 0.5 inch steriledisposable needle connected to a 50 μl glass Luer-hub Hamilton syringe.The site of injection is between L5 and L6. Post-procedural pain ismanaged with Ketoprofen (5 mg/kg, s.c.) at the time of IT injection, and24 to 48 hours later if the animal appears to be in discomfort.

Detection of RAN-Translated Peptides:

Cytoplasmic inclusions immunopositive for a poly(GP) antibody arepresent in the frontal cortex of C9orf72^(mutant) transgenic mice asshown in FIG. 9. To determine whether C9orf72^(mutant) transgenic miceexpress other RAN-translated peptides, and to evaluate whether theextent of RAN translation increases with age, expression of poly(GA),poly(GR) and poly(GP) peptides are examined at multiple time-pointsusing rabbit polyclonal antibodies. These antibodies, which specificallydetect their immunogen and show no cross-reactivity with other peptidesRAN-translated from sense or antisense transcripts of the C9orf72 repeatexpansion (FIG. 10A), detect neuronal inclusions throughout the CNS ofc9FTD/ALS patients (FIG. 10B).

At 2, 4, 8, and 12 months of age, brain and spinal cord are harvestedfrom wild-type and transgenic mice. Each brain is hemisected across thesagittal midline: one half is fixed in 10% formalin, while the otherhalf is dissected into 6 regions (cortex, subcortex, hippocampus,midbrain, brainstem and cerebellum) and frozen. Each spinal cord is cutinto 4 transverse sections; sections 1 and 3 are fixed, and sections 2and 4 are frozen.

For immunohistochemical studies, fixed spinal cord and hemi-brains areembedded in paraffin and sectioned (sagitally for brain and transverselyfor spinal cord). Sections are immunostained with poly(GA), poly(GR) andpoly(GP) antibodies using the DAKO Autostainer (DAKO Auto MachineCorporation) with DAKO Envision+ HRP System. In addition, to assess theextent to which inclusions of RAN translated peptides are found only inneurons, as is the case in human c9FTD/ALS brain,double-immunofluorescence staining is carried out using antibodies forRAN-translated peptides and neuronal or astrocytic makers.

To assess expression levels of RAN-translated peptides, and peptidesolubility changes in an age-dependent manner, frozen brain and spinalcord tissues are subjected to sequential extractions to collectfractions of soluble and insoluble proteins. These fractions areexamined by Western blot and quantitative electrochemiluminescentimmunoassay using poly(GA), poly(GR) or poly(GP) antibodies.

Effect of Anti-C9orf72 miRNAs on Expression of RAN-Translated Peptidesin C9orf72^(mutant) Transgenic Mice:

To assess the extent to which silencing of C9orf72 transcripts byrAAV.Rh10-C9mir decreases expression of poly(GP) peptides and other RANtranslated products expressed in C9orf72^(mutant) transgenic mice, brainand spinal cord of mice are harvested at various time-pointspost-transduction. IHC, Western blot and immunoassay analysis ofRAN-translated peptides are carried-out to the number of inclusions, aswell as levels of soluble and/or insoluble RAN-translated peptides.

TABLE 6 Summary of endpoints and outcome measures for animal studiesEndpoint/Procedure Histology GFP to track cellular distribution ofvector Chat/NeuN as a co-stain with GFP to track Neurons GFAP as aco-stain with GFP to track Astrocytes Cd11b as a co-stain with GFP totrack Microglia Olig1 as a co-stain with GFP to track OligodendricytesG₄C₂ FISH to assess RNA foci in the nucleus (FIG. 5) RAN-Translatedproteins to assess a RAN translation products (FIG. 9) mRNA, miR andVector Genome Quantification C9orf72 mRNA RT-qPCR are as shown in FIG. 2C9orf72 pre-mRNA RT-qPCR as shown in FIG. 4 C9-miR QuantificationRT-qPCR using custom assays rAAV Quantification qPCR for rAAV genomes(biodistribution)

Digital Image Analysis: Quantification RAN-Translated Protein Foci:

Analysis of IHC stained slides for RAN-translated proteins is performedusing the Aperio positive pixel count image analysis program. Wholeslides are scanned and digitized using Aperio Software. Analyses areconducted on the entire brain and spinal cord sections unless stainingartifacts are noted, such as precipitated chromogen. These areas areexcluded from analysis using the pen tool to outline the region. Theanalysis procedure is conducted on all images which are submitted forbatch processing using the Spectrum software. This process subjects allIHC stained slides to the one standard positive pixel count algorithm.The default settings used for brown chromogen quantification are in thethree intensity ranges (220-175, 175-100, and 100-0). Pixels which arestained, but do not fall into the positive-color specification, areconsidered negative stained pixels. These pixels are counted as well, sothat the fraction of positive to total positive and negative pixels isdetermined. Positivity (%) data are reported as the number of positivepixels (medium and strong positives)/total positive and negative pixelnumber.

Quantification of G₄C₂ Nuclear Foci:

The frequency of the RNA foci are assessed by transect-sampling acrossthe cerebellum and gray matter of the spinal cord. Microscopic fieldsare randomly chosen by a blinded operator and photographed with an oilimmersion 60× lens. Automated counting of RNA foci in the images is thencarried out using the FishJ algorithm macro in the ImageJ software.

Statistical Considerations:

Quantification of relative changes in gene expression of the C9orf72mRNA and pre-mRNA transcripts after AAV delivery with the variousconstructs are analyzed using the 2^(−ΔΔct) equation. To determinestatistical significant differences average values are compared for thecontrol groups (GFP-Scramble-miR) or (PBS controls) to averages from theexperimental group (AAVRh-C9-miR) using a two sample t-test forstatistical significance. Digital image analysis for RNA nuclear fociand RAN-translated proteins from transgenic mouse tissue sections arequantified by FishJ and Aperio software respectively. Values areobtained for each animal and averaged according to groups forstatistical analysis (Student t-test). The data for cerebellum andspinal cord from each group are analyzed individually.

Example 3: miRNA Targeting of C9orf72 in Primates

Assessment of rAAV.Rh10-C9miR and the Extent of Silencing of Transcriptsof C9orf72 in the Central Nervous System of Non-Human Primates (NHP)after Intrathecal Delivery.

The extent to which intrathecally administered rAAV.Rh10-C9miR spreadsthroughout spinal cord, cerebrum and cerebellum in non-human primates(NHPs) is assessed. Three rAAV.Rh10 vectors are prepared for thedelivery of expression constructs encoding anti-C9 miRNA. The vectorsare delivered via the intrathecal injection. Both spread and tropism ofAAVRh10 are assessed, as well as the silencing efficacy of C9-miRs fromtwo different promoters over a3 week period. One of the cohorts isinjected with vectors expressing the miRNA is from a polymerase IIpromoter (a hybrid chicken beta actin promoter) driving GFP. Anothercohort is administered a bicistronic vector in which the miRNA isexpressed from a U6 polymerase III promoter which is placed upstream ofthe hybrid chicken beta actin promoter driving GFP expression (FIG. 7B).A third cohort receives a GFP only control (see Table 7 below). RT-qPCRis performed on RNAs obtained from motor neurons that are laser capturedby micro-dissection. The dose of vector used is based on IT rAAVdelivery in NHP studies showing robust cortical and spinal cordtransduction.

TABLE 7 Short-term C9orf72 Silencing Study in NHPs Comparing Pol II vs.Pol III Promoters rAAV Construct CB6-GFP CB6-GFP-C9-miR U6-C9-miR-GFPNumber of Animals 3 Marmosets under 4 3 Marmosets under 4 3 Marmosetsunder 4 years of Age years of Age years of Age Route of Delivery andAAVRh10 Intrathecal AAVRh10 Intrathecal AAVRh10 Intrathecal Volume (300ul) (300 ul) (300 ul) Total rAAV Dose l × 10¹² vector 1 × 10¹² vector 1× 10¹² vector particles (vp) particles particles Approx. NHP Weight400-500 grams 400-500 grams 400-500 grams Approx. Dose/Weight 2 × 10¹²vp/Kilogram 2 × 10¹² vp/Kilogram 2 × 10¹² vp/Kilogram Promoter DrivingChicken/Beta Actin Chicken/Beta Actin Chicken/Beta Actin GFP PromoterDriving Polymerase II (same Polymerase II (same Polymerase III (U6 miRNAas above) as above) promoter)

rAAV.Rh10-C9-miR vectors that do not encode GFP are used to examinelong-term safety of viral delivery of rAAV.Rh10 in marmosets (e.g., 4controls and 4 treated). In-life endpoints include detailed physicalexaminations, detailed clinical observations, body weight, standardhematologic and chemistry parameters in blood, assessment of serumantibodies to AAVRh10, T cell responses to AAV peptides, and extent ofshedding of vector in body fluids and excreta. Post mortem endpointsinclude gross necropsy observations, organ weights and histopathology,and blood and tissue rAAV.Rh10-C9-miR vector DNA content (Table 9). Inaddition the extent of miR silencing of C9orf72 is assessed usingmethods disclosed herein.

TABLE 8 Endpoints for the NHP biodistribution/toxicology study. EndpointAssessment Timing of Endpoint Clinical assessment Daily QuantitativeTaqman PCR of blood Day 0, 1, 7, 21, 90 Quantitative Taqman PCR of semenDay 0, 1, 7, 21, 90 Quantitative Taqman PCR of multiple organs* Time ofsacrifice Necropsy with multiple organ* histopathology Time of sacrificeComplete Blood Counts (Hematocrit, Day 0, 1, 3, 7, 21, 90 leukocytes,platelets) Chemistry Panel (electrolytes, BUN, creatinine, Day 0, 1, 3,7, 21, 90 AST, ALT, CK) INF-Gamma ELISPOT for AAVRh10 capsid Day 0, 7,21, 60, 90 Neutralizing AAV Antibodies Day 0, 7, 21, 60, 90 Bisulfitesequencing for T_(reg) analysis Time of sacrifice (*the organ panel forhistopathology and quantitative PCR includes brain, spinal cord, heart,lungs, liver, kidney, spleen, pancreas, jejunum, gonads, muscle atinjection site, and inguinal lymph node)

Laser Capture Microdissection:

12 mm lumbar spinal cord frozen sections are collected onto PEN membraneslides (Zeiss, Munich, Germany) and stained with 1% Cresyl violet(Sigma, St. Louis, Mo.) in methanol. Sections are air dried and storedat −80° C. After thawing, motor neurons are collected within 30 min fromstaining using the laser capture microdissector PALM Robo3 Zeiss) usingthe following settings: Cut energy: 48, LPC energy: 20, Cut focus:80/81, LPC focus: 1, Position speed: 100, Cut speed: 50. About 500 MNsare collected per animal. Non-neuronal cells from the ventral horn arecollected from the same sections after collecting the motor neurons. RNAis then isolated using the RNaqueous Micro Kit (Ambion, Grand Island,N.Y.) according to manufacturer's instructions.

Interferon Gamma Elispot in Response to rAAV.Rh10 Capsid:

To characterize the immune response to rAAV.Rh10 after IT delivery,lymphocyte proliferation to pooled rAAV.Rh10 capsid peptides areassessed using the ELISPOT assay. Blood obtained at the various timepoints is processed using a standard Ficoll-Paque™ Plus protocol toobtain peripheral blood mononuclear cells. PBMC at a concentration of2×10⁵ cells per well is added to a plate coated with IFN-gamma captureantibody. Antigen specific stimulation with rAAV.Rh10 is performed for18-24 hrs after which cells are thoroughly washed. This is followed byaddition of the detection antibody and subsequently Avidin-HRP which isdeveloped with the appropriate substrate for a colorimetric reading.

AAV Neutralizing Antibody Assays:

The presence of AAV-Neutralizing antibodies are assessed usingappropriate techniques at a vector core laboratory.

Example 4: Assessment and Targeting of SOD1 Expression

Delivery of the rAAV Vector to Transgenic Mice

A recombinant adeno-associated viral vector has been developed thatdelivers miRNAs against SOD1 (See FIGS. 11A-C) to cells in vitro or invivo. Delivery of the rAAV vector to transgenic mice expressing themutant form of SOD1 resulted in 80-90% knockdown of the target mRNA intransduced tissues (FIG. 12). For example, it was determined anti-SOD1(miR) silences expression of SOD1 in mouse liver, as shown in FIG. 13.

For these experiments rAAV vectors are used with three types ofconstructs: (a) chicken beta actin (CB) driving GFP followed by tandemanti-SOD1 miRs (mir127); (b) the U6 promoter driving miR-SOD1 followedby CB-GFP; and as a control CB-GFP alone. FIG. 13 (top panel) showsschematics of these constructs.

It was determined that intrathecally delivered rAAV9 bearing a microRNAto attenuate expression of SOD1 prolongs survival in SOD1^(G93A)transgenic ALS mice. 2.4×10¹⁰ viral genomes/5 ul injected into thelumbar intrathecal space of 60 day old mouse achieved widespreaddelivery of the microRNA to multiple cell types along the spinal cord.(This dose is ˜ 1/16th the dose used in our IV delivery). In animalsthat were highly transduced, reduction of SOD1 expression by ˜50% wasobserved as assessed by western immunoblotting. Also observed was aprolongation of survival by ˜14 days overall and to more than 160 daysin mice with the highest level of SOD1 silencing (as compared to 123days in ALS mice treated with rAAV9-scrambled microRNA). These resultsindicate that C9-miR220 can be administered along the length of thespinal cord in rodents and non-human primates using rAAV.Rh10.

FIG. 20 indicates that treatment of G93A SOD1 mice with CB-miR-SOD1significantly increases survival compared to control animals. G93A SOD1mice were injected with 2×10¹² genome copies (gc) of CB-GFP orCB-miR-SOD1-GFP vector at day 56-68 of age and blindly monitored untiladvanced paralysis required euthanasia. Median survival was 108 days forcontrol animals (CB-GFP, n=19) and 130 days for CB-miR-SOD1-GFP (n=28).Log-rank test results in a p-value of 0.018, suggesting that increasedsurvival is statistically significant. These data further indicate thatsystemic delivery of mir-SOD1 by intravenous injection results insignificant increase in survival of G93A SOD1 mice.

Example 5: miRNA-Targeting of SOD1 in Primates

Intrathecal Delivery of Recombinant AAV (rAAV) Expressing SOD1 miRNA toBrain and Spinal Cord.

Adeno-associated virus (AAV)-mediated delivery of microRNA has been usedto silence SOD1 in mammalian tissues, including spinal cord.

As shown in FIG. 6 intrathecal administration of rAAV.Rh10.EGFP to anadult marmoset resulted in remarkable uptake in gray matter of both the(A) lumbosacral and (B) cervical anterior horns, with prominent labelingof motor neurons (as identified by their sizes and location).Laser-capture lumbo-sacral motor neurons were obtained from thismarmoset (treated with rAAV.Rh10.EGFP) and another treated withrAAV.Rh10.U6-miR-SOD1, which expresses a SOD1 silencing miR. As in FIG.6 (bottom), the control animal showed high levels of SOD1 transcript andminimal (essentially 0 baseline) miR-SOD1. By contrast, in the treatedanimal the SOD1 transcript was almost undetectable while there was ahigh level of the miR-SOD1 microRNA.

These constructs were used in studies conducted in nine marmosets, usingintrathecal delivery with a newer strain of AAV, designated AAV.Rh10.Details of the design are in FIG. 14. In certain instances, ITinjections in these monkeys resulted in tail flick and a dural “pop” asthe needle was inserted. In others, needle placement did not result inan observable tail flick. In both instances, there was good delivery ofAAV into the CSF. These points are illustrated in FIG. 15. Animals weresacrificed after 3.5-4 weeks. As the table in FIG. 15 notes, threeanimals were perfused with fixative (PFA) and six with saline. Three ofthe latter, which had the “good” injections, were used for laser captureof motor neurons and assays of miR and SOD1 levels in the motor neurons.

As shown in FIG. 16, after laser capture, MNs transduced with controlCB-GFP showed high levels of SOD1 and no miR-SOD1 as gauged by qPCR.Those transduced with U6-miR-SOD1 and CB-miR-SOD1 showed silencing ofSOD1 expression. The U6 construct produced more miR; in that animal,there was less SOD1 expression. FIGS. 17A-B extend the results to threeregions of the spinal cord (lumbar, thora and examines silencing in bothlaser-captured MNs and the residual tissue of the cord after MNs wereresected (non-MNs). There is evidence of SOD1 silencing in both sets oftissue. FIG. 18 shows silencing in the brainstem as assessed using qPCRwith tissue homogenates.

FIG. 19 uses RNA hybridization (“RNAScope”) to demonstrate in singlemotor neurons that expression of miR-SOD1 (in this case from the U6construct) correlates with absence of SOD1 expression, while in the MNtransduced with CB-GFP alone there is ample SOD1 expression. In summary,these data demonstrate that miR-SOD1 reagents are delivered to spinalcord after IT injection, and achieve substantial silencing of SOD1.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated that various alterations,modifications, and improvements will readily occur to those skilled inthe art. Such alterations, modifications, and improvements are intendedto be part of this disclosure, and are intended to be within the spiritand scope of the invention. Accordingly, the foregoing description anddrawings are by way of example only and the invention is described indetail by the claims that follow.

As used herein, the terms “approximately” or “about” in reference to anumber are generally taken to include numbers that fall within a rangeof 1%, 5%, 10%, 15%, or 20% in either direction (greater than or lessthan) of the number unless otherwise stated or otherwise evident fromthe context (except where such number would be less than 0% or exceed100% of a possible value).

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

The entire contents of all references, publications, abstracts, anddatabase entries cited in this specification are incorporated byreference herein.

1.-50. (canceled)
 51. A method of inhibiting expression of C9orf72 in asubject in need thereof, the method comprising administering to thesubject an effective amount of a synthetic microRNA (miRNA) that targetsa C9orf72 RNA transcript, wherein the synthetic miRNA specifically bindsto a nucleic acid sequence of the RNA transcript, wherein the nucleicacid sequence of the RNA transcript bound by the synthetic miRNA isencoded by a nucleic acid sequence as set forth in any one of SEQ ID NO:1, 3, 4, 5, or
 8. 52. The method of claim 51, wherein the syntheticmiRNA further comprises flanking regions of miR-155.
 53. The method ofclaim 52, wherein the RNA transcript is a pre-mRNA transcript.
 54. Themethod of claim 52, wherein the RNA transcript comprises a G₄C₂hexanucleotide repeat.
 55. The method of claim 53, wherein the pre-mRNAtranscript is a C9orf72 V₁ isoform transcript.
 56. The method of claim53, wherein the pre-mRNA transcript is a C9orf72 V₃ isoform transcript.57. The method of claim 53, wherein the pre-mRNA transcript is not aC9orf72 V₂ isoform transcript.
 58. The method of claim 51, wherein theRNA transcript comprises an intron.
 59. A synthetic microRNA (miRNA)that targets a C9orf72 RNA transcript, wherein the synthetic miRNAspecifically binds to a nucleic acid sequence of the RNA transcript,wherein the nucleic acid sequence of the RNA transcript bound by thesynthetic miRNA is encoded by a nucleic acid sequence as set forth inany one of SEQ ID NO: 1, 3, 4, 5, or 8, and wherein the synthetic miRNAfurther comprises flanking regions of miR-155.
 60. The synthetic miRNAof claim 59, wherein the RNA transcript is a pre-mRNA transcript. 61.The synthetic miRNA of claim 59, wherein the RNA transcript comprises aG₄C₂ hexanucleotide repeat.
 62. The synthetic miRNA of claim 60, whereinthe pre-mRNA transcript is a C9orf72 V₁ isoform transcript.
 63. Thesynthetic miRNA of claim 60, wherein the pre-mRNA transcript is aC9orf72 V₃ isoform transcript.
 64. The synthetic miRNA of claim 60,wherein the pre-mRNA transcript is not a C9orf72 V₂ isoform transcript.65. The synthetic miRNA of claim 59, wherein the RNA transcriptcomprises an intron.
 66. A recombinant nucleic acid encoding a syntheticmicroRNA (miRNA) that targets a C9orf72 RNA transcript, wherein thesynthetic miRNA specifically binds to a nucleic acid sequence of the RNAtranscript, wherein the nucleic acid sequence of the RNA transcriptbound by the synthetic miRNA is encoded by a nucleic acid sequence asset forth in any one of SEQ ID NO: 1, 3, 4, 5, or 8, and wherein thenucleic acid sequence encoding the synthetic miRNA is flanked by AAVinverted terminal repeats (ITRs).
 67. The recombinant nucleic acid ofclaim 66, further comprising a promoter operably linked with a regionencoding the synthetic miRNA.
 68. The recombinant nucleic acid of claim67, wherein the promoter is a tissue-specific promoter.
 69. Therecombinant nucleic acid of claim 67, wherein the promoter is an RNApolymerase II promoter
 70. The recombinant nucleic acid of claim 69,wherein the polymerase II promoter is a chicken β-actin (CBA) promoter.71. The recombinant nucleic acid of claim 67, wherein the promoter is anRNA polymerase III promoter.
 72. The recombinant nucleic acid of claim71, wherein the polymerase III promoter is a U6 promoter.
 73. Arecombinant adeno-associated virus (rAAV) comprising a recombinantnucleic acid encoding a synthetic miRNA that targets a C9orf72 RNAtranscript, wherein the synthetic miRNA specifically binds to a nucleicacid sequence of the RNA transcript, wherein the nucleic acid sequenceof the RNA transcript bound by the synthetic miRNA is encoded by anucleic acid sequence as set forth in any one of SEQ ID NO: 1, 3, 4, 5,or 8, and further comprising AAV inverted terminal repeats (ITRs). 74.The rAAV of claim 73, further comprising one or more capsid proteins ofone or more AAV serotypes selected from the group consisting of: AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11, andvariants thereof.
 75. The rAAV of claim 73, wherein the synthetic miRNAcomprises flanking regions of miR-155.
 76. The rAAV of claim 73, whereinthe RNA transcript is a pre-mRNA transcript.
 77. The rAAV of claim 73,wherein the RNA transcript comprises a G₄C₂ hexanucleotide repeat. 78.The rAAV of claim 73, wherein the RNA transcript comprises an intron.79. A composition comprising the rAAV of claim 73.